- Email: [email protected]
Pericruciate cortical neurons projecting to brain stem reticular formation, dorsal column nuclei and spinal cord in the cat
Neuros¢/enc¢ Letters, 1 (1975) 257--262
257
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
PERICRUCIATF~ CORTICAL NEURONS PROJECTD4G TO BRAIN STEM RETICULAR FORMATION, DORSAL COLUMN NUCLEI AND SPINAL CORD IN THE CAT
C.E. BERREVOETS and H.G.J.M. KUYPERS
Department of Anotomy, Erasmus University Medical School, Rotterdam (The Netherlands) (Received October 28th, 1975) (Accepted October 31st, 1975)
SUMMARY
Cells of origin of the pericruciate cortical fibers to the bulbar medial reticular formation, the dorsal column nuclei and the spinal cord in the cat were identified by means of the retrograde axonal t ~ a s p o r t of horseradish peroxidase. After injection of the enzyme in the dorsal column nuclei or the spinal cord many layer V pyramidal neurons were labeled retrogradely in areas 2, 3 and 4 (see ref. 6), but area 4 giant Betz cells were only l~beled after spinal cord injections. Medial bulbar reticular formation injections resulted in the labeling of pyramidal neurons mainly in area 6.
Anatomical studies [3,9,13,15,16] in the cat have shown that pericruciate cortical fibers are distributed to the sph~l cord, nuclei cuueatus and gracfiis and medial bulbar reticular formation. According to some of these studies [ 3, 9] fibers to the spinal cord and dors~A column nuclei come mainly from the lyre- and the postcruc~te gyri, while the bulk of the fibers to the bulbar reticular formation comes from a rostromedially adjoining area [9]. in the present investigation cells of origin of these projections were identified by utilizing the retrograde axonal transport of ho~:seradish peroxidase ~HRP) from fiber term'hmls and damaged axons to the parent cell b o d i ~ [7,8,11,14]. In three cats 30% HRP was injected in the nuclei cuneatus an~ gracflis on one side at six differen~ levels. In three other cats the injections were made only in the nucleus gz~-.ili~. In one additmnal cat one p~amida! tract was inten.upted two weeks pr'~r to the injection of the ipsilateral nucleus gracilis. Iv six cats the enzyme was injected unilaterally in the spinal cord, in three cats in the C1 segment and in three others in the L1 segment. The spinal injections were made in such a way that the needle penetrations damaged a great many fibers, in order to obtain enzyme transport through many cortical fibers [8] descending through these segments. In each of the spinal cords 3--4 transverse rows of closely spaced needle penetrations were made uni-
258
l a t e r ~ y through the white and the gray matter and every 0.25 mm along each needle p e n s i o n 0.2 #1 (to*~130~1) was deposited [12]. In two animals I ~130% HRP was i n j e c t ~ in the medial reticular formation at four different levels. The injection needle was introduced into the brain stem through its ventral surface, lateral to the pyramidal tract, and was advanced at an angle into the medial reticular formatiorL In nearly all expeTiments the Sigma VI HRP was injected, but in one ~pinal cord and in one nucleus gracflls the Boehringer enzyme was used, because it gave a relatively stronger accumulation of HRP reaction products in the neuronal cell bodies. Three days after the injection, the animals were perfused according ~) the method described earlier [ 1 2 ~ The injection sites were cut trarmversally in 40/ira frozen sections while the ~emispheres were cut in the sagittal plane. The sections were treated accordini~ to the method of GrahamKarnovsky [ 5] and some were counterst~ined wi~;h cresyl violet. The material was studied microscopically under bright, and dark-field illumination [ 11] and the location of the retrogradely labeled neurons in every fifth section was charted with the aid of an X-Y plotter. The cyto~chitectural rdbdivisions of the perieruciate cortex of Hauler and Mul~Clement [6] (Fig. 1A) was followed. After injections in the dorsal column nuclei considerable amounts of HRP reaction product were present in these nuclei and some also in adjoining structures. In the contralateral pericruciate cortex many labeled neurons were p r i n t in areas 3a and 3b and in area 4. A limited number of labeled neurons was also present contralaterally in area 2 and a very few in area 6 (Fig. 1B). The labeled neurons were generaUy of pyramidal shape and were :ocated mainly in lamina V. However, none of the giant Betz cells in area 4y were labeled after dorsal column inje~ions (Fig. 2B and D). After injections in both the nucleus gracilis and the nucleus cuneatus the population of labeled neurons extended laterally up the coronal sulcus (Fig. 1B), while after injections in the nucleus gracflis to the labeled neurons were present only medially (Fig. 1D). In all six cases some labeled neurons were also present in the medial parts of areas 3 and 4 of the ipsUateral hemisphere. These neurons may have been labeled mainly from fibers in the non-injected nucleus gracflis which frequently suffered collateral damage and also contained HRP reaction products. This would be in keeping with the fact that virtually no labeled neurons were present in the ipsilateral hemisphere of the one case in which the ipsilateral pyramidal tract leading to the non-injected dorsal column nuclei had been interrupted prior to the nvcleus graci!k injections. In the six cats with mdlateral spinal cord injections many labeled neurons were also present in the pericruciate cortex, almost exclusively contralaterally. They were located in area 4 as well as in area 3 and a few in area 2. After lumbar injections the labeled neurons were concentrated in the m e ~ l parts o~ these areas (Fig. 1E) as was the case after nucleus gracilis injections (Fig. 1D). However, after C1 injections (Fig. 1C) the population of labeled neurons extended laterally up to the coronal sulcus in roughly the same way
259 CYTOARCHITECTURE
CUI~. A N D
GRAC.
SPIN. SEGMENT
C1
.~7 . . . . t'-'~"- ".;.''-'."J~ '~,,
- ~ . ~ " :.~2:::::.~ :::::::::::::::::::::::::::::::::::
~-
• ,~..-.-.:.:.:.:.:.:.-
GRAC.
SPIN. SEGMENT L1
MED.
RET. F O R M
m°qm~QewJ~
"":::':':':":
,°:
Fig. 1. A: the cytoarchitectural subdivisions of the pericTuciate cortex in the cat after Hassler and Muhs-Clement [6]. B--F: schematic representation of the cortical areas cuntaining retrogradely labeled neurons after contralateral horseradish peroxidase injections in the dorsal column nuclei (B and D), the spinal cord (C and E) and the medial bu|bar reticular formation (F). The presence of labeled Betz cells is indicated by diamond~.
260
as L~ar combined cuneatus and grac~ injections(Fig. 1B). The labeled neurons after spinalinjectionswere also mainly pyramidal in shape and were concentrat~ in lamina V. However, in contrast to the findings after injections of ~ e dorml column nuclei, the spinalinjectionsalways resulted in the labeling c~ a great many B ~ z cellsin area 4y (Fig. 2 C and D). In addition, in som~ of the cases with spinalinjectionsitappeared that the labeled neurons in area 3 tended to be slightlylarger,on average, than the area 3 labeled neurons after dorsal column injections.
Fig. 2. Bright-field (A and C) and dark-field (B and D) photomicrographs of retrogradely labeled neurons iI, area 4y of the cat (mafnification × 140). A and B: labeled neurons after contralateral nucleus ~acilis injections. C and D: after contralateral spinal cord injections. Note that after spinal cord injections Betz cells are also labeled, while after nucleus gracilis injections only small cells are labeled.
In the two cases with bulbar injectionsa largeamount of H R P reaction product was present in the bulbar reticularformation, mainly medially. In addition, in one of the two cases some fibers,lateralin the pyramidal tract on the injected side,were evenly stained brown, which strongly suggests that they had transported HRP. In both cases many labeled neurons were present in area 6 of both hemispheres though contmlaterally there was almost twice as m a n y as ips~lateraUy. Some scattered neuron~ were also present in area 4
261 (Fig. 1F). In t h e one case in which fibers in the pyramidal tract on the injected side were stained, many neurons were also labeled in areas 3 and 4 of the ipsflateral hemisphere including giant Betz cells in area 4y. The present findings strongly suggest that the pericmciate cortical fibers to the spinal cord, dorsal c o l u m n nuclei and medial reticular formation are in part derived from different sets of cortical neurons. According to these findings, the neurons which distribute fibers to t h e medial reticular formation are located mainly in area 6 bilaterally, while those distributing fibers to spinal cord and dorsal column nuclei are situated primarily in areas 3 and 4 with a few in areas 2 and 6, mainly contralaterally. This is in agreement with earlier anatomical [3,9,10] and physiological [1,2,4] findings. From the present findings it can also be inferred t h a t the fibers to the spinal cord and dorsal column nuclei are in part derived from different sets of neurons, since at least the giant Betz cells in area 4y could only be labeled from the spin~ cord. Obviously, this inference is only valid when the lack of retrograde labeling of Betz cells after dorsal column nuclei injections did n o t result from the failure of possible Betz cell collaterals to the dorsal c o l u m n nuclei to transport detectable amounts of enzyme to the parent cell body. This possibility can not be ruled out. The idea t h a t the cortical fibers to the spinal cord and dorsal column nuclei are in part derived from different sets of neurons, is in accordance with the findings of Gordon and collaborators [ 2,4] who observed that those area 5 neurons which in their study could be invaded antidrornically from the dorsal column nuclei could not be invaded from the spinal cord. These observations are, in turn, in keeping with cur impression that in some cases the labeled neurons in area 3 after spinal ,:ord injections tended to be somewhat larger, on average, than after dorsal column injections. ACKNOWLEDGEMENT
This investigation was in part supported by Grant 13-46-08A and B of the Dutch Organization for Fundamental Research in Medicine (FUNGO, ZWO). REFERENCES 1 Armand, J., Padel, Y., and Smith, A.M., Somatotopical organization of the corticospinal tract in cat motor cortex, Brain Res., 74 (1974) 209--227. 2 Brecht, A., Gordon, G., and Powell, T.P.S., In preparation. 3 Chambers, W.W., and Liu, C.N. Cortico-spinal tract of the cat. An attempt to correlate the pattern of degeneration with deficits in reflex activity following neocorti~al lesions, J. comp. Neurol., 108 (1907) 23--55.
4 Gordon, G., and Miller, R., Identification of cortical cells projecting to the dorsal column nuclei of the cat~ Quart. J. exp. Physiol., 54 (1969) 85--98. 5 Graham, R.C., and Ksrnovsky, M.J., Glomeruhr permeability. Ultrastructural cytochemical studies using peroxidase and protein tracers, Exp. Med., 124 (1966) 1123--1134 6 Hassler, R., und Muhs-Clement, K., Architektonischer Aufbau des sensomvtorischen und parietalen Cortex der Katze, J. H~nforsch., 6 (1964) 377--420.
262
7 ~nsson, l(., and Olsson, Y., Eetrograde axonal transport of protein, Brain Res., 29 (1971) 363-365. 8 ~zistensson, K., ~ z i Oisson, Y., Retrograde transport of horseradish peroxidase in tra~ axo~. Time relationships between transport and induction of chromatolysis, Brain R~., 79 (197'4) 101--109. 9 Kuypers, ILG.J.M, An anatomical analysis of corticobulbar connexions to the ports and lower brain szem in the eat, J. Anat. (Lond.),92 (1958) 198-218. 10 Kuypers, H.G.J.M., Central cortical projections to motor and sensory cell groups, Brain, 83 (1960) 161--184. 11 Kuypers, H.G.J.M., Kievit, J., and Groen-Klevant, A.C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Res., 67 (1974) 211--218. 12 Kuypers, H.G.J.M., and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the eat, Neuroscience Letters, 1 (1975) 9--14. 13 Kuypers, H.G.J.M., and Tuerk, J.D., The distribution of the cortical fibers within the nucleus cuneatus and graeilis in the eat, J. Anat. (Lor~i.), 98 (1967) 143--162. 14 LaVaii, J.H., a~ut LaVail, M.M., Retrograde axonal transport in the central nervous system, Seien¢.;, 176 (1972) 1416--1417. 15 Rossi, G.F., an~ ~Jrodal, A.. Corticofugal fibers to the brain stem reticular formation. An experimental study in the cat, J. Anat. (Lond.), 90 (1956) 42--62. 16 Walberg, F., Corticofugal fibers to the nuclei of the dorsal columns. An experimental study in the e~t, Brain, 80 (1957) 273--287.
257
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
PERICRUCIATF~ CORTICAL NEURONS PROJECTD4G TO BRAIN STEM RETICULAR FORMATION, DORSAL COLUMN NUCLEI AND SPINAL CORD IN THE CAT
C.E. BERREVOETS and H.G.J.M. KUYPERS
Department of Anotomy, Erasmus University Medical School, Rotterdam (The Netherlands) (Received October 28th, 1975) (Accepted October 31st, 1975)
SUMMARY
Cells of origin of the pericruciate cortical fibers to the bulbar medial reticular formation, the dorsal column nuclei and the spinal cord in the cat were identified by means of the retrograde axonal t ~ a s p o r t of horseradish peroxidase. After injection of the enzyme in the dorsal column nuclei or the spinal cord many layer V pyramidal neurons were labeled retrogradely in areas 2, 3 and 4 (see ref. 6), but area 4 giant Betz cells were only l~beled after spinal cord injections. Medial bulbar reticular formation injections resulted in the labeling of pyramidal neurons mainly in area 6.
Anatomical studies [3,9,13,15,16] in the cat have shown that pericruciate cortical fibers are distributed to the sph~l cord, nuclei cuueatus and gracfiis and medial bulbar reticular formation. According to some of these studies [ 3, 9] fibers to the spinal cord and dors~A column nuclei come mainly from the lyre- and the postcruc~te gyri, while the bulk of the fibers to the bulbar reticular formation comes from a rostromedially adjoining area [9]. in the present investigation cells of origin of these projections were identified by utilizing the retrograde axonal transport of ho~:seradish peroxidase ~HRP) from fiber term'hmls and damaged axons to the parent cell b o d i ~ [7,8,11,14]. In three cats 30% HRP was injected in the nuclei cuneatus an~ gracflis on one side at six differen~ levels. In three other cats the injections were made only in the nucleus gz~-.ili~. In one additmnal cat one p~amida! tract was inten.upted two weeks pr'~r to the injection of the ipsilateral nucleus gracilis. Iv six cats the enzyme was injected unilaterally in the spinal cord, in three cats in the C1 segment and in three others in the L1 segment. The spinal injections were made in such a way that the needle penetrations damaged a great many fibers, in order to obtain enzyme transport through many cortical fibers [8] descending through these segments. In each of the spinal cords 3--4 transverse rows of closely spaced needle penetrations were made uni-
258
l a t e r ~ y through the white and the gray matter and every 0.25 mm along each needle p e n s i o n 0.2 #1 (to*~130~1) was deposited [12]. In two animals I ~130% HRP was i n j e c t ~ in the medial reticular formation at four different levels. The injection needle was introduced into the brain stem through its ventral surface, lateral to the pyramidal tract, and was advanced at an angle into the medial reticular formatiorL In nearly all expeTiments the Sigma VI HRP was injected, but in one ~pinal cord and in one nucleus gracflls the Boehringer enzyme was used, because it gave a relatively stronger accumulation of HRP reaction products in the neuronal cell bodies. Three days after the injection, the animals were perfused according ~) the method described earlier [ 1 2 ~ The injection sites were cut trarmversally in 40/ira frozen sections while the ~emispheres were cut in the sagittal plane. The sections were treated accordini~ to the method of GrahamKarnovsky [ 5] and some were counterst~ined wi~;h cresyl violet. The material was studied microscopically under bright, and dark-field illumination [ 11] and the location of the retrogradely labeled neurons in every fifth section was charted with the aid of an X-Y plotter. The cyto~chitectural rdbdivisions of the perieruciate cortex of Hauler and Mul~Clement [6] (Fig. 1A) was followed. After injections in the dorsal column nuclei considerable amounts of HRP reaction product were present in these nuclei and some also in adjoining structures. In the contralateral pericruciate cortex many labeled neurons were p r i n t in areas 3a and 3b and in area 4. A limited number of labeled neurons was also present contralaterally in area 2 and a very few in area 6 (Fig. 1B). The labeled neurons were generaUy of pyramidal shape and were :ocated mainly in lamina V. However, none of the giant Betz cells in area 4y were labeled after dorsal column inje~ions (Fig. 2B and D). After injections in both the nucleus gracilis and the nucleus cuneatus the population of labeled neurons extended laterally up the coronal sulcus (Fig. 1B), while after injections in the nucleus gracflis to the labeled neurons were present only medially (Fig. 1D). In all six cases some labeled neurons were also present in the medial parts of areas 3 and 4 of the ipsUateral hemisphere. These neurons may have been labeled mainly from fibers in the non-injected nucleus gracflis which frequently suffered collateral damage and also contained HRP reaction products. This would be in keeping with the fact that virtually no labeled neurons were present in the ipsilateral hemisphere of the one case in which the ipsilateral pyramidal tract leading to the non-injected dorsal column nuclei had been interrupted prior to the nvcleus graci!k injections. In the six cats with mdlateral spinal cord injections many labeled neurons were also present in the pericruciate cortex, almost exclusively contralaterally. They were located in area 4 as well as in area 3 and a few in area 2. After lumbar injections the labeled neurons were concentrated in the m e ~ l parts o~ these areas (Fig. 1E) as was the case after nucleus gracilis injections (Fig. 1D). However, after C1 injections (Fig. 1C) the population of labeled neurons extended laterally up to the coronal sulcus in roughly the same way
259 CYTOARCHITECTURE
CUI~. A N D
GRAC.
SPIN. SEGMENT
C1
.~7 . . . . t'-'~"- ".;.''-'."J~ '~,,
- ~ . ~ " :.~2:::::.~ :::::::::::::::::::::::::::::::::::
~-
• ,~..-.-.:.:.:.:.:.:.-
GRAC.
SPIN. SEGMENT L1
MED.
RET. F O R M
m°qm~QewJ~
"":::':':':":
,°:
Fig. 1. A: the cytoarchitectural subdivisions of the pericTuciate cortex in the cat after Hassler and Muhs-Clement [6]. B--F: schematic representation of the cortical areas cuntaining retrogradely labeled neurons after contralateral horseradish peroxidase injections in the dorsal column nuclei (B and D), the spinal cord (C and E) and the medial bu|bar reticular formation (F). The presence of labeled Betz cells is indicated by diamond~.
260
as L~ar combined cuneatus and grac~ injections(Fig. 1B). The labeled neurons after spinalinjectionswere also mainly pyramidal in shape and were concentrat~ in lamina V. However, in contrast to the findings after injections of ~ e dorml column nuclei, the spinalinjectionsalways resulted in the labeling c~ a great many B ~ z cellsin area 4y (Fig. 2 C and D). In addition, in som~ of the cases with spinalinjectionsitappeared that the labeled neurons in area 3 tended to be slightlylarger,on average, than the area 3 labeled neurons after dorsal column injections.
Fig. 2. Bright-field (A and C) and dark-field (B and D) photomicrographs of retrogradely labeled neurons iI, area 4y of the cat (mafnification × 140). A and B: labeled neurons after contralateral nucleus ~acilis injections. C and D: after contralateral spinal cord injections. Note that after spinal cord injections Betz cells are also labeled, while after nucleus gracilis injections only small cells are labeled.
In the two cases with bulbar injectionsa largeamount of H R P reaction product was present in the bulbar reticularformation, mainly medially. In addition, in one of the two cases some fibers,lateralin the pyramidal tract on the injected side,were evenly stained brown, which strongly suggests that they had transported HRP. In both cases many labeled neurons were present in area 6 of both hemispheres though contmlaterally there was almost twice as m a n y as ips~lateraUy. Some scattered neuron~ were also present in area 4
261 (Fig. 1F). In t h e one case in which fibers in the pyramidal tract on the injected side were stained, many neurons were also labeled in areas 3 and 4 of the ipsflateral hemisphere including giant Betz cells in area 4y. The present findings strongly suggest that the pericmciate cortical fibers to the spinal cord, dorsal c o l u m n nuclei and medial reticular formation are in part derived from different sets of cortical neurons. According to these findings, the neurons which distribute fibers to t h e medial reticular formation are located mainly in area 6 bilaterally, while those distributing fibers to spinal cord and dorsal column nuclei are situated primarily in areas 3 and 4 with a few in areas 2 and 6, mainly contralaterally. This is in agreement with earlier anatomical [3,9,10] and physiological [1,2,4] findings. From the present findings it can also be inferred t h a t the fibers to the spinal cord and dorsal column nuclei are in part derived from different sets of neurons, since at least the giant Betz cells in area 4y could only be labeled from the spin~ cord. Obviously, this inference is only valid when the lack of retrograde labeling of Betz cells after dorsal column nuclei injections did n o t result from the failure of possible Betz cell collaterals to the dorsal c o l u m n nuclei to transport detectable amounts of enzyme to the parent cell body. This possibility can not be ruled out. The idea t h a t the cortical fibers to the spinal cord and dorsal column nuclei are in part derived from different sets of neurons, is in accordance with the findings of Gordon and collaborators [ 2,4] who observed that those area 5 neurons which in their study could be invaded antidrornically from the dorsal column nuclei could not be invaded from the spinal cord. These observations are, in turn, in keeping with cur impression that in some cases the labeled neurons in area 3 after spinal ,:ord injections tended to be somewhat larger, on average, than after dorsal column injections. ACKNOWLEDGEMENT
This investigation was in part supported by Grant 13-46-08A and B of the Dutch Organization for Fundamental Research in Medicine (FUNGO, ZWO). REFERENCES 1 Armand, J., Padel, Y., and Smith, A.M., Somatotopical organization of the corticospinal tract in cat motor cortex, Brain Res., 74 (1974) 209--227. 2 Brecht, A., Gordon, G., and Powell, T.P.S., In preparation. 3 Chambers, W.W., and Liu, C.N. Cortico-spinal tract of the cat. An attempt to correlate the pattern of degeneration with deficits in reflex activity following neocorti~al lesions, J. comp. Neurol., 108 (1907) 23--55.
4 Gordon, G., and Miller, R., Identification of cortical cells projecting to the dorsal column nuclei of the cat~ Quart. J. exp. Physiol., 54 (1969) 85--98. 5 Graham, R.C., and Ksrnovsky, M.J., Glomeruhr permeability. Ultrastructural cytochemical studies using peroxidase and protein tracers, Exp. Med., 124 (1966) 1123--1134 6 Hassler, R., und Muhs-Clement, K., Architektonischer Aufbau des sensomvtorischen und parietalen Cortex der Katze, J. H~nforsch., 6 (1964) 377--420.
262
7 ~nsson, l(., and Olsson, Y., Eetrograde axonal transport of protein, Brain Res., 29 (1971) 363-365. 8 ~zistensson, K., ~ z i Oisson, Y., Retrograde transport of horseradish peroxidase in tra~ axo~. Time relationships between transport and induction of chromatolysis, Brain R~., 79 (197'4) 101--109. 9 Kuypers, ILG.J.M, An anatomical analysis of corticobulbar connexions to the ports and lower brain szem in the eat, J. Anat. (Lond.),92 (1958) 198-218. 10 Kuypers, H.G.J.M., Central cortical projections to motor and sensory cell groups, Brain, 83 (1960) 161--184. 11 Kuypers, H.G.J.M., Kievit, J., and Groen-Klevant, A.C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Res., 67 (1974) 211--218. 12 Kuypers, H.G.J.M., and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the eat, Neuroscience Letters, 1 (1975) 9--14. 13 Kuypers, H.G.J.M., and Tuerk, J.D., The distribution of the cortical fibers within the nucleus cuneatus and graeilis in the eat, J. Anat. (Lor~i.), 98 (1967) 143--162. 14 LaVaii, J.H., a~ut LaVail, M.M., Retrograde axonal transport in the central nervous system, Seien¢.;, 176 (1972) 1416--1417. 15 Rossi, G.F., an~ ~Jrodal, A.. Corticofugal fibers to the brain stem reticular formation. An experimental study in the cat, J. Anat. (Lond.), 90 (1956) 42--62. 16 Walberg, F., Corticofugal fibers to the nuclei of the dorsal columns. An experimental study in the e~t, Brain, 80 (1957) 273--287.